Hairless triggers reactivation of hair growthby promoting Wnt signalingGerard M. J. Beaudoin III*†, Jeanne M. Sisk*, Pierre A. Coulombe‡§, and Catherine C. Thompson*†¶�

*Kennedy Krieger Research Institute and Departments of †Neuroscience, ¶Molecular Biology and Genetics, ‡Biological Chemistry, and §Dermatology,Johns Hopkins University School of Medicine, Baltimore, MD 21205

Communicated by Donald D. Brown, Carnegie Institution of Washington, Baltimore, MD, August 31, 2005 (received for review August 9, 2005)

The mammalian hair cycle involves periodic regeneration of a tinyorgan, the hair follicle, through a stem-cell-mediated process. TheHairless (Hr) gene encodes a nuclear receptor corepressor (HR) thatis essential for hair follicle regeneration, but its role in this processis unknown. Here, we demonstrate that transgenic expression ofHR in progenitor keratinocytes rescues follicle regeneration inHr�/� mice. We show that expression of Wise, a modulator of Wntsignaling, is repressed by HR in these cells, coincident with thetiming of follicle regeneration. This work links HR and Wnt func-tion, providing a model in which HR regulates the precise timing ofWnt signaling required for hair follicle regeneration.

corepressor � nuclear receptor � skin

Hair is maintained through a cyclic process that includesperiodic regeneration of hair follicles in a stem-cell-

dependent manner. The hair cycle consists of three definedstages: growth (anagen), followed by regression (catagen) andrest (telogen) (Fig. 1A) (1). Growth of a new hair requiresreentry into anagen, a process involving activation of multipotentepithelial stem cells residing in a specialized part of the follicleouter root sheath (ORS) known as the bulge (Fig. 1B) (2–4).Activating signals emanate from adjacent mesenchymal cells(dermal papilla), directing epithelial stem cells to migrate anddifferentiate to regenerate the hair bulb (Fig. 1B), the structurefrom which a new hair will emerge (2–5). Multiple signalingpathways, including Wnts, Sonic hedgehog (Shh), and TGF-�family members have been shown to promote anagen initiation,yet the exact mechanism by which hair follicles regenerate is notclear (5–7).

Disruption of Hairless (Hr) gene function causes a complexskin phenotype that includes a specific defect in hair follicleregeneration in both humans and mice (8–10). In Hr mutantmice, hair follicle morphogenesis and initial hair growth isnormal. However, after the follicles regress (catagen) and thehair is shed, around postnatal day (P)17, telogen stage folliclesnever reenter anagen, and no new hair is produced, resultingin alopecia (9, 11). Lack of hair regrowth has been attributedto separation of the bulge and dermal papilla (DP) duringcatagen (3, 12). Alternatively, the defect in anagen initiationmay ref lect a loss of the relevant epithelial stem cell populationor an inability to generate and�or interpret the necessarysignal(s).

We have shown that the Hairless protein (HR) is a nuclearreceptor corepressor and, therefore, acts by regulating geneexpression (13–15). Molecular studies of the epidermal compo-nent of the Hr mutant phenotype revealed that HR represses thetranscription of genes involved in epidermal differentiation andthat misregulation of these genes underlies the formation ofabnormal epidermal structures (utricles) (16). Although HRlikely regulates gene expression in hair follicle regeneration aswell, the role of HR in hair follicles is not known. Here, weaddress the role of HR in follicle regeneration and provideevidence that HR regulates this process by repressing theexpression of a Wnt inhibitor at the proper time in the hair cycle.

Materials and MethodsMouse Lines. Hr�/� mice and TOP-Gal mice have been de-scribed in refs. 16 and 17. K14-rHr transgenic mice were madeby cloning the coding sequence of the rHr cDNA downstreamof a human K14 promoter (18, 19) and were generated by theTransgenic Core Facility of the Johns Hopkins Medical Insti-tutions. Animal care was in accordance with institutionalguidelines.

Histology, Immunostaining, and in Situ Hybridization. Histologicalanalysis was as described in ref. 16. For immunofluorescencestaining for HR, frozen sections (8 or 20 �m) were fixed in 10%neutral-buffered formalin, blocked and permeabilized, and in-cubated with HR-specific antisera (1:250) (20). Anti-rabbitCy3-coupled antibody (1:2,000, Jackson Immunoresearch) wasused to detect HR antiserum. For subsequent detection of K14,sections were blocked in 1 �g�ml rabbit IgG and incubated withFITC-coupled rabbit anti-K14 antibody (1:500; Covance). Cellnuclei were visualized with DAPI (4�,6-diamidino-2-phenylin-dole) (Molecular Probes).

For in situ hybridization, frozen sections (20 �m) were hy-bridized with digoxygenin-labeled cRNA probes, as described inref. 15. Plasmids used to make probes were as follows: Shh,pBS-shh (16); Wise, IMAGE clone 5066413 (American TypeCulture Collection); Axin2, IMAGE clone 6827741; Scd1,IMAGE clone MGC-6427. For immunohistochemistry to detectHR protein, endogenous peroxidase activity was blocked, andsections were incubated with HR antisera (1:250). HR antibodywas detected with biotinylated goat anti-rabbit antibody followedby VECTASTAIN Elite ABC kit (Vector Laboratories) andDAB (3,3�-diaminobenzidine) (Sigma).

For alkaline phosphatase staining to identify DP, frozensections (20 �m) were fixed in neutral-buffered formalin, stainedwith 4-nitroblue tetrazolium chloride�5-bromo-4-chloro-3-indolyl-phosphate and counterstained with Nuclear Fast red(Vector Laboratories). �-gal was detected in frozen sections (20�m), as described in refs. 17 and 21. Subsequent immunostainingfor HR protein was as described above.

Photomicrographs were prepared by digital picture captureand processed with Adobe PHOTOSHOP, as described in refs. 13and 16. Hair follicles were staged by examination of folliclemorphology as described in ref. 22.

RNA and Protein Analysis. Protein preparation and Westernanalysis were as described in ref. 16. Total RNA was preparedfrom back skin from one male and one female for eachgenotype with Trizol (Sigma). Northern analysis and quanti-fication was as described in refs. 16 and 23. Probes wereprepared from cDNAs for Wise and �-tubulin [kindly providedby A. Lanahan (Dartmouth University, Hanover, NH)]. Wise

Abbreviations: DP, dermal papilla; ORS, outer root sheath; Pn, postnatal day n; Shh, sonichedgehog.

�To whom correspondence should be addressed at: Kennedy Krieger Institute, 707 NorthBroadway, Baltimore, MD 21205. E-mail: thompsonc@kennedykrieger.org.

© 2005 by The National Academy of Sciences of the USA

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(24) was also identified as sclerostin-domain-containing gene1 (Unigene: 25), sclerostin-like (26), ectodermal bone mor-phogenetic protein inhibitor (Ectodin) (27) and USAG-1 (28).

Wise was not cited in previous microarray results becausemisexpression was not significant at P12 (�2-fold) (16).

Cell Culture and Transfection Assays. To isolate the promoter of theWise gene, the Vista Genome Browser (http:��pipeline.lbl.gov�cgi-bin�gateway2?bg � mm4&selector � vista) was used toidentify conserved regions between mouse and human genomicsequences. PCR of mouse genomic DNA with gene-specificoligonucleotides was used to isolate a fragment (�1430 to �65),which was cloned into pGL2-Basic (Promega); reporter geneactivity was increased �100-fold compared with the promoter-less vector.

Expression plasmids for rHR and silencing mediator of reti-noid and thyroid hormone receptors (SMRT) (kindly providedby R. Evans, Salk Institute) were described in refs. 13 and 29.TR-interacting domains (TR-ID) (13) are mutated in mtHR;TR-ID1 overlaps the site of vitamin D receptor binding (15).Expression plasmids for Wnt1 and 3a were kindly provided by J.Nathans (Johns Hopkins School of Medicine). The Wnt 10bexpression plasmid was made by using a partial Wnt10b cDNA[kindly provided by G. Shackleford (University of SouthernCalifornia, Los Angeles)] and RT-PCR; the coding sequencewas cloned into pCMX.

The GC cell line [kindly provided by P. Mellon (University ofCalifornia, San Diego)], which supports HR corepressor activity,was grown in DMEM with 10% horse serum and 5% FBS. SuperTOP-Flash (STF) cells (kindly provided by Dr. J. Nathans) are293 cells that stably express a reporter gene with seven lymphoidenhancer factor�T cell-specific-factor (LEF�TCF)-binding sitesdriving luciferase expression (30). STF cells were grown inDMEM: F12 with 10% FBS.

For GC transfections, cells were plated in 24-well plates andtransfected with 150 ng of reporter plasmid, 50–75 ng ofexpression plasmid and 150 ng of RSV-�gal by using Lipo-fectamine 2000 (Invitrogen). Cells were harvested in passive lysisbuffer (Promega) and assayed for �-gal and luciferase activity.Luciferase activity was normalized to �-gal activity to correct fortransfection efficiency. Data are the average of four experimentsdone in duplicate. For STF transfections, cells were transfectedwith 75 ng of each expression plasmid and 25 ng of CMV-�galby using Lipofectamine 2000. Cells were processed as for GC;data are the average of three experiments done in duplicate.

Results and DiscussionTo determine where and when HR acts in hair follicles, welocalized nuclear HR protein throughout the hair cycle usingHR-specific antisera. We find that follicles actively growing hairin anagen (P12) do not contain detectable HR (Fig. 1C). HRprotein is detected as follicles enter catagen (P17) (Fig. 1C),which coincides with the onset of phenotypic alterations in hairfollicles of Hr mutant mice (12, 16). Within the follicles, HRprotein is found in the nuclei of keratin 14 (K14)-positive cellsin the ORS, which includes the bulge region (Fig. 1 C–E). HRis also detected in K14-negative hair bulb cells but not in the DP(Fig. 1 C and D). HR expression is maintained in the ORSthrough late catagen into telogen and the early part of the nextanagen (Fig. 1F). Once the hair bulb has reformed in mid-anagen, HR protein is again undetectable (Fig. 1F). Thus, HRprotein expression is spatially and temporally regulated duringthe hair cycle, consistent with function of HR in both follicleregression and anagen reinitiation.

Because HR protein is localized to K14-positive ORS cellsduring the rest and reinitiation phases of the hair cycle, wepredicted that expression of HR in this subset of progenitorkeratinocytes would be sufficient to initiate the first postnatalanagen and rescue hair growth in Hr�/� mice. Therefore, weattempted to rescue the hair phenotype of Hr�/� mice by creatingmice that express HR only in progenitor keratinocytes. First, we

Fig. 1. HR protein expression in hair follicles during the hair cycle. (A) Schematicrepresentation of the hair cycle depicting changes in follicle structure. (B) Sche-matic of an anagen-stage hair follicle; relevant structures are indicated. A and Bwere adapted from ref. 5. (C–F) Immunofluorescent detection of HR protein (red)and keratin 14 (K14) (green). DAPI staining (blue) indicates nuclei. (C) HR proteinexpression in mouse back skin at P12 and P17. HR is detected in wild type (���)hair folliclesatP17; thesignal is specificbecausethere isnostaininginHr�/� (���)follicles (Right). DP is denoted by white dots. Boxed areas are magnified below.(D) Magnification of region in white box shows HR staining in nuclei (HR�DAPI)and in K14-positive ORS and K14-negative hair bulb (HR�K14). Arrowheadsdenote nuclear staining. (E) Magnification of region in red box shows HR stainingin K14-positive ORS. (F) HR expression through the hair cycle. Shown is immuno-fluorescent staining for HR (red) and K14 (green). Hair cycle stages are indicated.Arrowheads indicate HR signal. (Scale bars, 50 �m.)

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generated transgenic mice that constitutively express HR inprogenitor keratinocytes by using the K14 promoter (K14-rHr).In K14-rHr skin, HR protein expression is high early in postnataldevelopment (P9, P12) and remains above wild type levels asdevelopment proceeds (Fig. 2A). Analysis of the K14-rHr phe-notype showed shorter hair (Fig. 2B), associated with decreasedproliferation of matrix cells (data not shown). Thicker epidermis(Fig. 2C) is attributed to an expansion of the undifferentiated

compartment (shown by K14 staining) and a decrease in therelative number of terminally differentiated keratinocytes(shown by filaggrin staining), suggesting that epidermal differ-entiation is delayed. Sebaceous differentiation is similarly de-layed, because sebaceous glands are not detected at P9 (Fig. 2D)but are visible in some follicles at P12 (data not shown).Together, these results are consistent with our model that HRnormally promotes differentiation toward hair cell fate andsuppresses or delays differentiation into epidermis and seba-ceous glands (16).

To generate mice that express HR only in K14-positive cells(‘‘transgenic rescue’’), we crossed the K14-rHr transgenic micewith Hr�/� mice (Fig. 3 A and B). At P22, both Hr�/� andtransgenic rescue mice exhibit hair loss, consistent with histo-logical analysis that shows prominent utricles and clusters of cellsin the subdermal fat layer (Fig. 3C). Despite the prior defect, thetransgenic rescue mice subsequently regenerate hair and even-tually display thick fur (Fig. 3B). By P28, the transgenic rescuehair follicles resemble wild type follicles (Fig. 3C) and expressShh mRNA, a marker of early anagen (6) (Fig. 3D, arrowheads),in contrast to Hr�/� mouse skin, which remains grossly abnormal(Fig. 3C) and does not express Shh (Fig. 3D) (16). Thus, HRexpression in K14-expressing cells rescues the ability of Hr�/�

hair follicles to regenerate via the normal pathway. Failure torescue hair loss may be due to a function of HR in K14-negativecells (Fig. 1D). The location of the DP is similar in Hr�/� andtransgenic rescue skin (Fig. 3E), indicating that separation of theDP and bulge is not responsible for lack of hair regrowth andsupporting the idea that a diffusible signal between DP and bulgereinitiates hair growth (3).

The demonstration that HR function in progenitor keratino-cytes is sufficient for hair follicle regeneration led us to inves-tigate whether HR regulates Wnt signaling, because activation ofthis pathway in these cells has been directly implicated in hairfollicle regeneration (31–35). We examined expression of aWnt-responsive gene, Axin2 (36–38), and found that Axin2expression is not detected in Hr�/� hair follicles at anageninitiation (Fig. 4A). Remarkably, Axin2 expression is restored intransgenic rescue skin (Fig. 4A). Because HR is a corepressor,we hypothesized that HR may promote Wnt activation byrepressing the expression of Wnt inhibitor(s). From a previousmicroarray screen for HR-regulated genes, we noted that thereis, indeed, a potential Wnt inhibitor that is slightly up-regulatedin Hr�/� skin at P12, before hair loss (16). This gene encodesWise (Wnt modulator in surface ectoderm), a protein that hasbeen shown to modulate Wnt signaling (24). To play a role in hairfollicle regeneration, at the time of anagen reinitation, Wiseexpression should (i) increase in Hr�/� skin, (ii) decrease intransgenic rescue skin (relative to Hr�/�), and (iii) repress Wntsignaling in hair progenitor keratinocytes. Northern analysisconfirmed that Wise is up-regulated (5.1-fold) in Hr�/� skin andreduced 2-fold (relative to Hr�/�) in transgenic rescue skin (Fig.4B). Notably, expression is also reduced (1.7-fold) in K14-rHrmice, consistent with overexpression of a repressor and suggest-ing that HR represses Wise expression.

To determine whether the Wise gene is repressed by HR,genomic regions containing the promoter for Wise were clonedupstream of a luciferase reporter gene, and activity was mea-sured in the absence and presence of HR. Expression of HRsignificantly reduced activity of the Wise reporter gene (Fig. 4C).Repression is specific, because HR did not affect the activity ofa minimal thymidine kinase promoter (Fig. 4C). Additionally,expression of another nuclear receptor corepressor, silencingmediator of retinoid and thyroid hormone receptors (SMRT)(39) had no effect on activity (Fig. 4C). The Wise promoterregion contains putative binding sites for nuclear receptors thatinteract with HR, including thyroid hormone receptors (TRs),vitamin D receptor (VDR), and retinoid orphan receptors

Fig. 2. Transgenic expression of HR in progenitor keratinocytes. (A) Westernanalysis showing HR protein expression in wild type (WT) and K14-rHr (TG)mouse skin at the indicated postnatal ages. Detection of �-tubulin (Lower)shows protein loading. Molecular mass markers are indicated in kDa. (B)Hematoxylin–eosin staining of sections from TG and wild type (���) back skinat P9. (Scale bar, 100 �m). (C) (Upper) Hematoxylin–eosin staining of TG andwild type (���) epidermis. (Bottom) Immunofluorescent detection of K14(green, arrows) and filaggrin (red, arrowheads). DAPI (blue) staining indicatesnuclei. (D) In situ hybridization for Scd1, a sebaceous gland marker, in TG andwild type (���) back skin at P9. Signal is detected only in wild type (arrow-heads). (Scale bar, 20 �m in C and D.)

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Fig. 3. Hr expressed by the K14 promoter rescues hair regrowth in Hr�/� skin.(A) Immunofluorescent detection of HR protein (red) and K14 (green) in Hr�/�

(���), transgenic rescue (���;T), and wild type (���) back skin. DAPI (blue)staining indicates nuclei. (Scale bar, 50 �m.) (B) Mice of the indicated geno-types at 7 weeks. (C) Hematoxylin–eosin staining of back skin sections from theindicated ages in Hr�/� (���), transgenic rescue (���;T), and wild type (���)mice. Arrows indicate cells in the dermis of Hr�/� and transgenic rescue miceat P22. Arrowheads indicate reformed hair bulbs in transgenic rescue and wildtype mice at P28. *, cyst in Hr�/� at P28. (Scale bar, 100 �m.) (D) In situhybridization detecting Shh mRNA in P28 mouse back skin. Shh expression(arrowheads) is detected in transgenic rescue (���;T) and wild type (���)hair follicles. Black dots outline follicle remnant in Hr�/� mice (���). (Scalebar, 20 �m.) (E) Alkaline phosphatase staining localizing the DP (arrows) inHr�/� (���), transgenic rescue (���;T), and wild type (���) back skin at P22.Brackets approximate position of the bulge; asterisks indicate club hair.Sections are counterstained with Nuclear Fast red. (Scale bar, 20 �m.)

Fig. 4. HR regulates expression of a modulator of Wnt signaling (Wise).(A) In situ hybridization for Axin2, a Wnt-responsive gene, in back skinsections from P22 mice of the indicated genotypes: ��� (Hr�/�), ���;T(transgenic rescue), and ��� (wild type). Arrowheads indicate Axin2 ex-pression. (Scale bar, 20 �m.) (B) Northern analysis for Wise mRNA expres-sion using RNA from back skin of P24 mice of the indicated genotypes: ���(wild type); T (K14-rHr); ���, (Hr�/�); and ���;T (transgenic rescue).(Right) �-tubulin hybridization of an identical blot; Wise expression wasnormalized to �-tubulin expression. 18S, position of 18S RNA. (C) Reportergenes for the Wise promoter (Wise-Luc) and control (tk-Luc) were trans-fected into cells with the indicated expression vectors. (�), empty expres-sion vector. Results were normalized to vector control for each promoter.(D) The Wise reporter gene was transfected with expression vectors for HRor a HR derivative (mtHR) that has mutated receptor-interaction domains.Equal expression of HR and mtHR was verified by Western analysis (data notshown). (E) Cells stably expressing a Wnt-responsive reporter gene (SuperTOP-Flash) were transfected with the indicated expression vectors. For B–D,relative light units (RLU) is luciferase activity relative to �-gal activity(internal control). Results are the average of at least three experimentsdone in duplicate.

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(13–15). Consistent with repression by nuclear receptors, a HRderivative with mutations in TR- and VDR-binding domains nolonger represses the Wise promoter (Fig. 4D).

Considering the hypothesis that repression of Wise by HRregulates hair follicle regeneration requires demonstration thatWise functions as a Wnt inhibitor in this context. Using a cell linethat stably expresses a Wnt-responsive reporter gene (Super-TOP-Flash) (30), we find that expression of Wise dramaticallyinhibits Wnt1-induced reporter gene activity (20-fold) (Fig. 4E).Although the Wnt family member that directs hair follicleregeneration is not known, Wnt 10b is expressed at the time(anagen initiation) and place (follicle epithelial cells) to play arole in hair follicle regeneration (40). We find that Wnt 10b-induced reporter gene activity is reduced 28-fold by expressionof Wise (Fig. 4E). Notably, the cells used for this assay expressFrizzled-1 and Frizzled-7 (41), Wnt receptors that are found inhair follicles (42), suggesting that Wnt 10b activation in STF cellsrecapitulates signaling in vivo. Consistent with Wise function asa context-dependent regulator (24), Wise potentiated activationby Wnt3a, a Wnt not expressed at anagen initiation (40). Theseresults show that Wise can inhibit signaling by the Wnt (10b)most likely to play a role in anagen initiation.

If repression of Wise expression by HR regulates hair follicleregeneration, the appearance of nuclear HR in hair folliclesshould coincide with both decreased Wise expression and in-creased Wnt-dependent gene expression. To address this hy-pothesis, we first combined immunohistochemistry for HR pro-tein with in situ hybridization for Wise (Fig. 5 A and B). In lateanagen, when HR protein is undetectable, Wise mRNA isexpressed throughout the hair bulb (Fig. 5A). As follicles pro-ceed into early catagen, nuclear HR protein is detected at thebase of the bulb, and Wise mRNA expression becomes limitedto the upper part of the bulb. In late catagen, Wise expressionhas almost disappeared and remains absent from cells expressing

HR as follicles enter the next growth phase (Fig. 5B, earlyanagen). By mid-anagen, HR protein is no longer detected,whereas Wise mRNA expression is restored in the bulb andputative bulge (Fig. 5B). The inverse timing of HR protein andWise mRNA expression in the same cell population suggests thatHR is suppressing Wise expression in vivo, allowing hair cycleprogression.

Consistent with HR repressing Wise expression, Wise mRNAis present in Hr�/� skin at telogen, a stage at which Wiseexpression is undetectable in both wild type and transgenicrescue skin (Fig. 5C). Wise mRNA is localized to the lowerportion of the follicle remnant (Fig. 5C, arrowhead) and indermal cells (Fig. 5C, arrow). The follicle remnant and dermalcells express K14 and correspond to the location of new hairformation, thereby linking abnormally persistent expression ofWise with cells unable to reform a hair follicle (16, 43).

To test the prediction that nuclear HR protein is required forWnt-dependent gene expression in hair follicles, we analyzed HRexpression in the skin of TOP-Gal transgenic mice, which expressa �-gal reporter gene in response to Wnt��-catenin activation(17). Similar to previous reports, we detect �-gal expression incells adjacent to the club hair in the putative bulge region at thetelogen–anagen transition (Fig. 5D) (17, 32). Strikingly, we findthat �-gal activity coincides with nuclear HR protein in theputative bulge region (Fig. 5E). The significance of HR-expressing cells that lack �-gal activity is not clear but may reflecta role in modulating other signals. Subsequent to this stage, bothHR protein and �-gal activity are undetectable (data not shown).Thus, Wnt��-catenin activation is correlated with the presenceof nuclear HR protein at anagen initiation and inversely corre-lated with Wise mRNA expression.

Based on these data, we propose a model in which HRpromotes Wnt activation and, thus, hair growth initiation byrepressing expression of a soluble Wnt inhibitor at the proper

Fig. 5. HR expression is inversely correlated with Wise mRNA expression and directly correlated with Wnt activation in the bulge. (A) In situ hybridization forWise mRNA (blue, arrowheads) and immunohistochemistry for HR protein expression (brown, arrows) in P17 mouse back skin; follicles are at the indicated stages.Asterisks indicate pigment. (Scale bar, 50 �m.) (B) Expression of Wise mRNA (blue) and HR protein (brown) in P22 back skin; follicles are at the indicated stages.Brackets indicate bulge region; asterisks indicate club hair. (Scale bar, 50 �m.) (C) In situ hybridization for Wise mRNA in P24 mouse back skin. Expression isdetected in the follicle remnant (arrowhead) and dermal cells (arrow) in Hr�/� skin (���). Sense control probe with adjacent Hr�/� section (���, sense); asteriskindicates club hair. (Scale bar, 20 �m.) (D) Detection of �-gal reporter gene expression (blue, arrowhead) in TOP-Gal mouse back skin. Bright-field (Left) anddifferential interference contrast (Right) microscopy images. (Scale bar, 20 �m.) (E) Detection of reporter gene expression (blue, arrowhead) and immunohis-tochemical staining for HR (brown). HR protein colocalizes with Wnt��-catenin activation (blue) in the bulge. (Scale bar, 10 �m.)

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time in the hair cycle. This work links Hr gene function and Wntsignaling and provides a molecular mechanism for regulating theprecise temporal and spatial localization of Wnt signaling re-quired for hair-cycle reinitiation (33, 34, 44). Presumably, HRfunction is not needed during initial hair morphogenesis, be-cause undifferentiated ectoderm is not subject to the influenceof Wnt inhibitors, or an alternative factor may regulate Wntsignaling in this context.

Although it has been proposed that lack of follicle regenera-tion in Hr mutant mice is due to separation of the DP and bulge,we find that the position of the DP is similar in Hr�/� andtransgenic rescue skin (Fig. 3 C and E). Instead, the datadescribed here support the hypothesis that loss of Hr functionresults in the inability to generate or interpret a diffusible signal.Thus, our model includes a molecular mechanism to account forthe failure of hair follicles to regenerate in Hr mutant skin:inhibition of Wnt signaling caused by the persistent expressionof Wise and, potentially, other Wnt inhibitors.

Through its restricted expression and corepressor function,HR controls the timing and location of hair follicle regeneration.

Although the connection between HR and Wnt signaling isstrong, hair follicle regeneration is a complex process requiringcoordination of multiple signaling pathways, including Shh andBMPs (45). Molecular mechanisms underlying potential cross-talk between signaling pathways during hair follicle regenerationare not known. Notably, Wise has also been shown to functionas a modulator of BMP signaling (26–28). It is likely that HRregulates the expression of multiple genes important for follicleregeneration, which may include modulators of other signalingpathways.

We thank J. Zarach for preliminary data and resources, the Coulombelaboratory for support, C.-M. Fan (Carnegie Institution of Washing-ton) for TOP-Gal mice, Q. Xu and J. Nathans (Johns Hopkins Schoolof Medicine) for reagents and advice, and G. Seydoux and K. Smithfor critical reading of the manuscript. This work was supported byNational Institutes of Health Grants AR44232 (to P.A.C.) andNS41313 (to C.C.T.) and National Research Service Award NS44744(to G.M.J.B.).

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